Internal organs to be subjected to transplantation include, for example, liver. Liver is the largest parenchymatous organ in a human body. It has various functions such as bilirubin metabolism, drug metabolism, and blood coagulation factor production, typically including metabolisms of carbohydrates, proteins and lipids, playing a very important role in an organism.
Thus, severe hepatic failure is very dangerous for the life of a patient even if it is temporary. On the other hand, if a liver function can be substituted for about one week, due to highly regenerative ability of liver, a patient with liver damage caused by fulminant liver failure recovers. For such a serious liver disease, hepatic transplantation is the most effective therapy. However, not all the patient can receive its favor due to serious donor shortage. Under the current actual conditions in Japan, although the life-saving rate has been raised to some extent by a combination of continuous filtration dialysis and plasma exchange as the means for temporarily substituting a liver function, the rate is still insufficient (see, Abe, et al., “Study of plasma exchange for liver failure: beneficial and harmful effects.”, The Apher Dial, 2004, 8, p. 180-184), and the establishment of more effective treatment methods have been eagerly desired.
Under such circumstances, therapeutic artificial livers are highly expected, especially, development of bioartificial livers filled with cells in order to utilize a metabolic ability and a protein synthesis ability of living cells is attracting attention. A bioartificial liver is a module prepared by incorporating and fixing hepatocytes in carriers, and it can be said as an artificial liver device simulating liver in human body. The blood in a subject is introduced in the device and the removal of harmful substances in the blood and the feed of biologically active substances such as coagulation factor derived from the liver cells can be carried out by utilizing the metabolizing ability of hepatocytes.
In one example of the experiments of human hepatocytes culturing by the present inventors, the donor was a white male (56 years old) who had experienced head trauma by a traffic accident and the cause of his death was subarachnoid hemorrhage. The transport time from US was about 39 hours. Hepatocytes were separated by normograde perfusion using a collagenase and then, cold-preserved in William's Medium E, and air-transported from Chiba prefecture to Okayama prefecture. The hepatocytes were cultured in culture medium comprising mainly William's Medium E to which deleted form hepatocyte growth factor (dHGF) was added at various concentrations of 0 ng/ml for group A), 10 ng/ml for group B), 100 ng/ml for group C) and 1000 ng/ml for group D), and the effects of dHGF were assessed based on a growth ability by the MIT assay; metabolic abilities of ammonia, lidocaine and diazepam, and an albumin production ability.
In the dHGF-added groups, cell growth was significantly better. Metabolic abilities of ammonia, lidocaine and diazepam and an albumin production ability were significantly better in the groups B) and C), while in the group D), 1000 ng/ml dHGF addition, a drug metabolic ability and an ammonia production ability per given cell number lowered. Visual observation also showed that a cobblestone morphology of hepatocytes was maintained well in the groups B) and C).
In Europe and the United States, hepatocytes are separated from the liver unsuitable for transplantation, and adopted clinically to hepatocyte transplantation and bioartificial livers. However, in Japan, the liver unsuitable for transplantation is prescribed as incineration and thus, it cannot be used for the bioartificial livers. Therefore, a donor liver judged to be unsuitable for transplantation (because of reasons such as fatty liver and intense fibrosis) in the United States was obtained from National Disease Research Interchange (NDR1) via HUMAN & ANIMAL BRIDGING RESEARCH ORGANIZATION laboratory (Ichikawa city, Chiba prefecture, responsible person: Dr. Satoshi Suzuki) in the form of liver block (130 g) which was then separated into liver cells, and a functional cultivation method thereof was investigated. It is extremely important to develop a cultivation method aiming at functional maintenance of human hepatocytes separated from such donor liver unsuitable for transplantation that is only usable resource.
As a countermeasure for the problem wherein use of healthy human hepatocytes is impossible, there have been trials of induction into hepatocytes from human peripheral blood stem cells, myeloid stem cells and liver precursor cells. These cells, however, show poor growth ability and thus, it is not realistic to obtain sufficient number of cells necessary for application to bioartificial livers (at least one billion). Therefore, in Europe and China, clinical trials of bioartificial livers using porcine hepatocytes have been carried out in humans (see, van de Kerkhove, et al., “Phase I clinical trial with the AMC-bioartificial liver.”, Academic Medical Center Int J Artif Organs, 2002, 25, p. 950-959, Donini, et al., “Temporary neurological improvement in a patient with acute or chronic liver failure treated with a bioartificial liver device”, Am J Gastroentrol, 2000, 95, p. 1102-1104, Mazariegos, et al., “Safety observations in phase I clinical evaluation of the Excorp Medical Bioartificial Liver Support System after the first four patients.”, ASAIO J, 2001, 47, p. 471-475, Ding, et al., “The development of a new bioartificial liver and its application in 12 acute liver failure patients.”, World J Gastroenterol, 2003, 9, p. 829-832, Demetriou, et al., “Prospective, randomized, multicenter, controlled trial of a bioartificial liver in treating acute liver failure.”, Ann Surgery, 2004, 239, p. 660-670, Mundt, “A method to assess biochemical activity of liver cells during clinical application of extracorporeal hybrid liver support.”, Int J Artif Organs, 2002, 25, p. 542-548, Morsiani, et al., “Early experiences with a porcine hepatocyte-based bioartificial liver in acute hepatic failure patients.”, Int J Artif Organs, 2002, 25, p. 192-202, Xue, et al., “TECA hybrid artificial liver support system in treatment of acute liver failure.”, World J Gastroenterol, 2001, 7, p. 826-829).
It is expected that cell or tissue culture technologies can be adopted industrially for regenerative medicine, cell preparation, useful substance production (bioreactor), investigation and research into function of tissue, organ and internal organ, screening of new drugs, animal experiment substitute methods for evaluating influences of endocrine disrupting chemicals, and cell chips, typically including cell transplantation and bioartificial organs.
Conventionally, as a method for culturing animal cells having adhesiveness, a two-dimensional cultivation method, that is, a so-called monolayer cultivation method has been generally used, in which a substrate such as a culture dish made of polystyrene or glass is used and the surface thereof is coated with a living body-derived factor, or treated chemically or physicochemically, and cells are adhered to and spread on the surface. For example, if cells are cultured on a polystyrene dish coated with collagen, an animal-derived intercellular matrix component, or on a polystyrene dish having a surface hydrophilized by plasma treatment, the cells adhere to and spread on the surface, thereby taking a cell morphology in which cytoplasm is spread in flat form.
On the other hand, cells isolated from tissue and internal organs of organisms, so-called primary cells, often maintain properties and functions of tissue and internal organs from which the cells are originated and thus, these cells have a great deal of potential in industrial application. However, it is known that in the monolayer cultivation method, properties and functions of various cells will be lost in several days or several weeks, in most cases. Particularly, in the case of primary hepatocytes which are well-differentiated and have various complicated functions among primary cells, properties and functions thereof tend to be lost quickly in the monolayer culture. For example, it is known that if hepatocytes isolated from rat liver are monolayer-cultured, important functions of liver, that is, a protein synthesis function, a detoxification function and a drug metabolic function are lowered or lost within several days from initiation of culture. It is hypothesized that in a monolayer cultivation method, cells have cytoplasm in the form of flat two-dimensional state and thus, mechanisms originally possessed by cells in a living body, such as an intracellular structure, polarity, and information exchange due to bonding with adjacent cells, are lowered and lost, causing lowering and loss of properties and functions originally possessed by cells (see, Japanese Unexamined Patent Publication No. 128660/2001).
In order to avoid such lowering and loss of the properties and functions originally possessed by cells, a so-called “three-dimensional cultivation method” has drawn attention in which cells are mutually assembled to construct a three-dimensional structure similar to living tissue. Scaffolds in such a three-dimensional cultivation method are roughly classified into two types. One is an animal-derived intercellular matrix component, and another is a synthetic polymer. Examples of the animal-derived intercellular matrix component include collagen gel, laminin, and animal basement membrane-derived component (trade name: Matrigel, available from Becton Dickinson and Company (constituents: laminin 56%, collagen IV 31% and entactin 8%)). It is reported that when, for example, rat hepatocytes are cultured with Matrigel, spheroid is formed (see, Bissell, et al., “Transcriptional regulation of the albumin gene in cultured rat hepatocytes. Role of basement-membrane matrix.”, Mol Biol Med, 1990, 7, p. 187-197). Examples of the synthetic polymer include polyglucosic acid and poly L-lactic acid. It is reported that when, for example, rat hepatocytes are cultured with polyglucosic acid, spheroid is formed (see, Fiegel, et al., “Influence of flow conditions and matrix coatings on growth and differentiation of three-dimensionally cultured rat hepatocytes.”, Tissue Eng, 2004, 10, p. 165-174).
Since cells cultured by the three-dimensional cultivation method are capable of maintaining properties and functions originally possessed by cells at higher level for a longer period of time as compared with cells cultured by a two-dimensional cultivation method, as described above, it is anticipated that the cells cultured by a three-dimensional cultivation method can be highly effective means for industrial applications such as bioartificial organs, regeneration medicine, cell preparation, useful substance production (bioreactor), investigation and research of function of tissue, organ and internal organ, screening of new drugs, animal experiment substitute methods for evaluating influences of endocrine disrupting chemicals, and cell chips.